Bioactive compound

ABSTRACT

This invention relates to a bioactive compound and to compositions which contain it. The invention further relates to methods of microtubule stabilization. In particular, it relates to a compound which has cytotoxic properties and which therefore has utility in inter alia anti-tumor treatments.

[0001] This invention relates to a bioactive compound and tocompositions which contain it. The invention further relates to methodsof microtubule stabilization. In particular, it relates to a compoundwhich has cytotoxic properties and which therefore has utility in interalia anti-tumor treatments.

BACKGROUND

[0002] In the search for anti-cancer drugs, compounds from naturalsources, such as paclitaxel, extracted from the bark of the Pacific yewtree, have displayed useful anti-cancer activity and proven successfulin clinical trials.

[0003] Marine sponges of the genus Mycale (Carmea) are a rich source ofbioactive secondary metabolites of diverse structures. The mycalysines,mycalolides, deoxytedanolide and the macrolide pateamine have all beenisolated from members of this genus and exhibit a variety of properties,including cytotoxic properties. See, for example, Perry et al., J. Am.Chem. Soc. (1988), 110, 4850-4851 and Northcote et al., TetrahedronLetters (1991) 32, 6411-6414.

[0004] The strategy of using tubulin as a target for cancer chemotherapyis based on the increased growth and division of cancer cells and thefact that drugs that interfere with mitosis such as the vinca alkaloidsthat depolymerize microtubules have proven effective in the treatment ofcancer. Paclitaxel (Taxol®) and taxotere (Docetaxel®) target tubulinbut, unlike the vinca alkaloids and colchicine, cause polymerization andstabilization of microtubules. Both are currently used therapeuticallyfor the treatment of solid tumors of the breast, ovary, and lung (He L.,Orr G. A., Horwitz S. B., Drug Discovery Today (2001), 6, 1153-1164).Microtubule-stabilizing compounds can be divided into three groups:

[0005] (a) diterpenes, including the taxanes, paclitaxel and taxotere,isolated from Yew trees (He L. et al.) and eleutherobin/sarcodictyin,isolated from marine corals (Long B. H., Carboni J. M., Wasserman A. J.,Cornell L. A., Casazza A. M., Jensen P. R., Lindel T., Fenical W.,Fairchild C. R., Cancer Res. (1998), 58, 1111-1115);

[0006] (b) macrolides, including epothilones, isolated from thebacterium Sorangium cellulosum (Bollag D. M., McQueney P. A., Zhu J.,Hensens O., Koupal L., Liesch J., Goetz M., Lazarides E., Woods C. M.,Cancer Res. (1995), 55, 2325-2333 and Kowalski R. J., Giannakakou P.,Hamel E., J. Biol. Chem. (1997), 272, 2534-2541) and laulimalides,isolated from the marine sponge Cacospongia mycofijiensis (Mooberry S.L., Tien G., Hernandez A. H., Plubrukarn A., Davidson B. S., Cancer Res.(1999), 59 653-660); and

[0007] (c) polyhydroxylated alkatetraene lactones, includingdiscodermolide, isolated from a Caribbean sponge (Ter Haar E., KowalskiR. J., Hamel E., Lin C. M., Longley R. E., Gunasekera S. P., RosenkranzH. S., Day B. W., Biochem. (1996), 35, 243-250 and Kowalski R. J.,Giannakakou P., Gunasekera P., Longley R. E., Day B. W., Hamel E.,Molec. Pharmacol. (1997), 52, 613-622).

[0008] The complex chemical syntheses required to produce clinicallyuseful amounts of such drugs has limited their development asanti-cancer agents, although both epothilone and the more complexpaclitaxel and taxotere have now been synthesized in sufficient amountsfor clinical use. In addition, paclitaxel is lipophilic, thus having lowaqueous solubility, and for clinical use, it must be dissolved inCremaphor/ethanol, a vehicle that contributes to paclitaxel'sundesirable side effects that include hypersensitivity reactions,neutropenia, peripheral neuropathy, and alopecia (Bollag D. M. et al.).Paclitaxel's hydrophobicity also promotes the acquisition of themultiple drug resistance (MDR) phenotype through expression ofP-glycoprotein (P-gp) (Parekh H., Wiesen K., Simpkins H., Biochem.Pharmacol. (1997), 53, 461-470). P-gp is responsible for the efflux of abroad range of organic solutes from the cell, and paclitaxel is just oneof these. In addition toover-expression of P-gp, some cells becomeresistant as a result of mutation of the paclitaxel binding site onβ-tubulin (Giannakakou P., Gussio R., Nogales E., Downing K. H.,Zaharevitz D., Bollbuck B., Poy G., Sackett D., Nicolaou K. C., Fojo T.,Proc. Nat. Acad. Sci. (USA) (2000), 97, 2904-2909).

[0009] Therefore there is a need for other microtubule-stabilizers withsimilar anti-mitotic activity to paclitaxel but which lack theinteraction with P-gp or which bind to unique sites on the tubulinpolymer. Epothilones, laulimalides, and discodermolides have shownpromise in this area, displaying less loss of toxicity to certainP-gp-expressing cells than paclitaxel (Bollag D. M. et al., Kowalski R.J. et al., Mooberry S. L. et al. and Kowalski R. J. et al.), althoughstill being transported to some extent by P-gp. At least three of theknown microtubule-stabilizing drugs, the epothilones (Bollag D. M. etal. and Kowalski R. J. et al.), discodermolide (Kowalski R. J. et al.),and the eleutherobins (Long B. H.), compete with [³H]-paclitaxel for itsbinding site on β-tubulin; however, epothilone and discodermolide alsoshow different sensitivities to particular β-tubulin mutations despitebinding to a similar site. The paclitaxel binding site of β-tubulin isavailable at 3.5 Å resolution (Nogales E. et al.), facilitating drugmodeling approaches. A common pharmacophore has been partiallydescribed, but further structure/function studies are needed (He L. etal., Giannakakou P. et al., He L., Jagtap P. G., Kingston D. G. I., ShenH -J., Orr G. A., Horwitz S. B., Biochem. (2000), 39, 3972-3978 andNicolaou K. C., Ritzén A., Namoto K., Chem. Comm. (2001), 17,1523-1535).

[0010] Recent in vivo tests on tumor formation in nude mice have shownpromise for desoxyepothilone analogues, specificallyZ-12,13-desoxyepothilone B (dEpoB) and its more water soluble analogue,dEpoF (Chou T. -C., O'Conner O. A., Tong W. P., Guan Y., Zhang Z. -G.,Stachel S. J., Lee C., Danishefsky S. J., Proc. Natl. Acad. Sci. (USA)(2001), 98, 8113-8118). Interestingly, the parent compound, epothiloneB, although more potent than dEpoB or dEpoF, is too cytotoxic in vivofor use as an anti-cancer drug.

[0011] The applicants have now identified a bioactive compound from amarine sponge of the genus Mycale. The applicants have established thecompound as a novel microtubule-stabilizing agent with potentiallyunique properties to the other known microtubule-stabilizing drugs. Itis towards this compound, which the applicants have termed Peloruside A,to its functionally equivalent analogues, and to compositions, uses andmethods of treatment which employ these compounds, that the presentinvention is broadly directed.

SUMMARY OF THE INVENTION

[0012] In a first aspect, the invention therefore provides a compound offormula (I);

[0013] wherein R₁, R₂, R₃, R₄ and R₅ are independently hydrogen, alkylor acyl; or a functionally equivalent analogue thereof.

[0014] In a further aspect, the invention provides a compound of formula(II);

[0015] wherein X is O or ═C(R₅)R₆ and R₁, R₂, R₃,R₄, R₅, and R₆ areindependently hydrogen, alkyl or acyl; or a functionally equivalentanalogue thereof.

[0016] Preferably, the compound is of formula (III);

[0017] or a functionally equivalent analogue thereof.

[0018] In another aspect, the invention provides a compound of formula(IV);

[0019] or a functionally equivalent analogue thereof.

[0020] In another aspect, the invention provides a bioactive compoundwhich has the NMR and/or IR spectral signature of FIGS. 1 and 2.

[0021] In another aspect, the invention provides composition whichcomprises a compound of the invention together with a suitable carriertherefor.

[0022] Preferably, the composition is a pharmaceutical composition.

[0023] In another further aspect, the invention provides a method ofprophylaxis or therapy which comprises the step of administering to apatient in need of the same a compound or a composition of theinvention.

[0024] Preferably, the prophylaxis or therapy is achieved by inhibitingthe proliferation of cells.

[0025] Preferably, the compound is administered in an amount effectiveto provide microtubule stabilization.

[0026] A preferred method is a treatment of a patient against cancer.

[0027] The above formulae specify relative stereochemistry only.

DESCRIPTION OF THE DRAWINGS

[0028] While the invention is broadly as described above, it will alsobe appreciated that it is not limited thereto but also includesembodiments of which the following description provides examples. Inparticular, a better understanding of the present invention will begained through reference to the accompanying drawings in which:

[0029]FIG. 1 shows the ¹H NMR spectral signature for Peloruside A;

[0030]FIG. 2 shows the IR spectral signature for Peloruside A;

[0031]FIG. 3 shows the structures of Peloruside A, the NaBH₄ reductionproduct of Peloruside A, Epothilone A, Laulimalide and Paclitaxel;

[0032]FIG. 4 shows (2A) H441 cells, (2B) Peloruside A treated H441 cellsand (2C) paclitaxel treated H441 cells;

[0033]FIG. 5 shows the progression of H441 cells for a control against 1μM Paclitaxel and 1 μM Peloruside; and

[0034]FIG. 6 shows for HL-60 cells immunoblotting for differentconcentrations of Peloruside, paclitaxel, the NaBH₄ reduction product ofPeloruside, and colchicine

DESCRIPTION OF THE INVENTION

[0035] As described above, the present invention has as its primaryfocus a new bioactive compound and its functionally equivalentanalogues. This compound has been isolated from a marine sponge of thegenus Mycale from Pelorus Sound, New Zealand. It has also been found,inter alia, to have cytotoxic properties; hence the name Peloruside A.

[0036] The compound of the invention can be isolated from marine spongesobtained from New Zealand coastal waters (including Pelorus Sound,HalfMoon Bay, Stewart Island and Kapiti). The sponges are a specieswhich belongs to the genus Mycale (Family Mycalidae, OrderPoecilosclerida). Individuals of this species may be encrusting ormassive, with a chocolate brown ectosome, often with a purple tinge. Thesponge surface often has large oscules (2-4 mm diameter) and may appearstippled due to the presence of polychaete worm tubes. The choanosome islight brown with a reticulate skeleton composed of tracts ofsubtylostyles (220-270 mm long) interspersed with microscleres:anisochelae of 2 size classes, 18-20 and 26-30 mm; sigmas, 20-26 mm; andraphides. The skeleton at ectosome consists of spicules identical to thechoanosome, but tangentially arranged and supported by erect spiculebrushes.

[0037] Sponge specimens which contain Peloruside A can be readilycollected manually, generally at depths of 3 to 20 meters, during thewinter months.

[0038] Such sponges can be farmed commercially should this providedesirable.

[0039] A variety of methods can be used to isolate and purify PelorusideA from samples of Mycale, including solvent extraction, partitionchromatography, silica gel chromatography, liquid-liquid distribution ina Craig apparatus, adsorption on resins, and crystallization fromsolvents.

[0040] The isolation and purification methods chosen can be monitored ateach step by performing in vitro and/or in vivo antitumor tests asdescribed by Geran R. I., Greenberg N. H., MacDonald M. M., SchumacherA. M. and Abbott B. S. in Cancer Chemother. Rep. (1972), Part 3, 3 (2),1-103, and by Schmidt J. M. and Pettit G. R., in Experientia (1978), 34,659-660. Such tests include the determination of the concentration ofactive material required to inhibit the growth of tumor cells in culture(eg. the concentration required to inhibit growth by 50 percent or theE.D.₅₀) and of the dose of active material required to prolong the lifeof mice bearing transplanted tumors.

[0041] A preferred extraction process is described in the examples.

[0042] Peloruside A has the structure set out in formula (III) above.However, analogues and/or structural variants of Peloruside A whichretain substantially equivalent bioactivity to Peloruside A also formpart of the invention. For example, any of the accessible OH groupsshown in the formulae can be replaced with, for example, alkyl groupsprovided that the poly-oxygenation of the subject molecule overall isnot significantly reduced. Equally, the methoxy groups can be replacedwith OH groups or longer chain alkoxy groups.

[0043] The selection of substituent groups and the processes by whichtheir substitution can be achieved will be a matter of routine choicefor the skilled worker in this field.

[0044] Further variations target the alkene side chain, with the lengthof the chain being altered.

[0045] The alkene side chain present in the compounds of formula (I) maybe derivatized to prepare compounds of formula (II). By the use ofozonolysis or other suitable techniques known to persons skilled in theart, the alkene carbon-carbon double bond may be cleaved to give themethyl ketone represented by formula (II), wherein 'X is O. This methylketone may be derivatized by the use of the Wittig reaction, or othersuitable synthetic reaction such as are well-known to those skilled inthe art, to give the compounds represented by formula (II), wherein X is═C(R₅)R₆, wherein R₅ and R₆ are independently hydrogen, alkyl or acyl.

[0046] Analogues within the scope of the invention will retain themacrolide structure, inclusive of the pyranose ring and gem-dimethyls asshown in Formula (III).

[0047] The fact that Peloruside A has free hydroxyl groups also meansthat acyl esters can be prepared. Such acyl esters of Peloruside A canbe prepared by methods well known to those skilled in the art. Acylderivatives of Peloruside A can be used for the same biological purposesas the parent compound.

[0048] Acids which can be used in the acylation of Peloruside A to formcompounds of formula (I) include:

[0049] (d) saturated or unsaturated, straight or branched chainaliphatic carboxylic acids, for example, acetic, propionic, butyric,isobutyric, tert-butylacetic, valeric, isovaleric, caproic, caprylic,decanoic, dodecanoic, lauric, tridecanoic, myristic, pentadecanoic,palmitic, margaric, stearic, acrylic, crotonic, undecylenic, oleic,hexynoic, heptynoic or octynoic acid;

[0050] (e) saturated or unsaturated, alicyclic carboxylic acids, forexample, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid,cyclopentenecarboxylic acid, methylcyclopentenecarboxylic acid,cyclohexanecarboxylic acid, dimethylcyclohexanecarboxylic acid ordipropylcyclohexanecarboxylic acid;

[0051] (f) saturated or unsaturated, alicyclic aliphatic carboxylicacids, for example, cyclopentaneacetic acid, cyclopentanepropionic acid,cyclohexaneacetic acid, cyclohexanebutyric acid ormethylcyclohexaneacetic acid;

[0052] (g) aromatic carboxylic acids, for example, benzoic acid, toluicacid, naphthoic acid, ethylbenzoic acid, isobutylbenzoic acid ormethylbutylbenzoic acid; and

[0053] (h) aromatic-aliphatic carboxylic acids, for example,phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamicacid, phenylpropiolic acid and naphthylacetic acid, and the like.

[0054] Suitable halo-, nitro-, hydroxy-, keto-, amino-, cyano-,thiocyano-, and lower alkoxyhydrocarbon carboxylic acids includehydrocarboncarboxylic acids as given above which are substituted by oneor more of halogen, nitro, hydroxy, keto, amino, cyano, or thiocyano, orlower alkoxy, advantageously lower alkoxy of not more than six carbonatoms, for example, methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy,and isomeric forms thereof.

[0055] As described below, Peloruside A has been determined to havecytotoxic properties in tests which are predictive of cytotoxic(including anti-tumor) activity in mammals, including humans. Theapplicants have further determined that like paclitaxel, Peloruside Aarrests cells in the G₂/M phase of the cell cycle and induces apoptosis.

[0056] Such properties therefore render Peloruside A suitable for use,alone or together with other active agents, in a number of therapeuticapplications, including in anti-tumor treatments. In addition, therelatively simple structure of Peloruside A makes it suitable for thedesign and synthesis of analogues with improved tumor targeting andreduced tumor cross-resistance.

[0057] The administration of Peloruside A is particularly useful fortreating animals or humans bearing a neoplastic disease, for example,acute myelocytic leukemia, acute lymphocytic leukemia, malignantmelanoma, adenocarcinoma of the lung, neuroblastoma, small cellcarcinoma of the lung, breast carcinoma, colon carcinoma, ovariancarcinoma, bladder carcinoma, and the like.

[0058] The dosage administered will be dependent upon the identity ofthe neoplastic disease, the type of host involved, age, health, weight,kind of concurrent treatment, if any, frequency of treatment andtherapeutic ratio.

[0059] Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.1 to about 200 mg/kg;intraperitoneal, 1 to about 500 mg/kg; subcutaneous, I to about 500mg/kg; intramuscular, 1 to about 500 mg/kg; orally, 5 to about 1000mg/kg; intranasal instillation, 5 to about 1000 mg/kg; and aerosol, 5 toabout 1000 mg/kg of animal (body) weight.

[0060] Expressed in terms of concentration, an active ingredient can bepresent in the compositions of the present invention for localized useabout the cutis, intranasally, pharyngolaryngeally, bronchially,broncholially, intravaginally, rectally, or ocularly in a concentrationof from about 0.01 to about 50% w/w of the composition; preferably about1 to about 20% w/w of the composition; and for parenteral use in aconcentration of from about 0.05 to about 50% w/v of the composition andpreferably from about 5 to about 20% w/v.

[0061] The compositions of the present invention are preferablypresented for administration to humans and animals in unit dosage forms,such as tablets, capsules, pills, powders, granules, suppositories,sterile parenteral solutions or suspensions, sterile non-parenteralsolutions or suspensions, and oral solutions or suspensions and thelike, containing suitable quantities of an active ingredient.

[0062] For oral administration either solid or fluid unit dosage formscan be prepared.

[0063] Powders are prepared quite simply by comminuting the activeingredient to a suitably fine size and mixing with a similarlycomminuted diluent. The diluent can be an edible carbohydrate materialsuch as lactose or starch. Advantageously, a sweetening agent or sugaris present as well as a flavouring oil.

[0064] Capsules are produced by preparing a powder mixture ashereinbefore described and filling into formed gelatin sheaths.Advantageously, as an adjuvant to the filling operation, a lubricantsuch as a talc, magnesium stearate, calcium stearate and the like isadded to the powder mixture before the filing operation.

[0065] Soft gelatin capsules are prepared by machine encapsulation of aslurry of active ingredients with an acceptable vegetable oil, lightliquid petrolatum or other inert oil or triglyceride.

[0066] Tablets are made by preparing a powder mixture, granulating orslugging, adding a lubricant and pressing into tablets. The powdermixture is prepared by mixing an active ingredient, suitably comminuted,with a diluent or base such as starch, lactose, kaolin, dicalciumphosphate and the like. The powder mixture can be granulated by wettingwith a binder such as corn syrup, gelatin solution, methylcellulosesolution or acacia mucilage and forcing through a screen. As analternative to granulating, the powder mixture can be slugged, i.e., runthrough the tablet machine and the resulting imperfectly formed tabletsbroken into pieces (slugs). The slugs can be lubricated to preventsticking to the tablet-forming dies by means of the addition of stearicacid, a stearic salt, talc or mineral oil. The lubricated mixture isthen compressed into tablets.

[0067] Advantageously the tablet can be provided with a protectivecoating consisting of a sealing coat or enteric coat of shellac, acoating of sugar and methylcellulose and polish coating of camauba wax.

[0068] Fluid unit dosage forms for oral administration such as syrups,elixirs and suspensions can be prepared wherein each teaspoonful ofcomposition contains a predetermined amount of active ingredient foradministration. The water-soluble forms can be dissolved in an aqueousvehicle together with sugar, flavouring agents and preservatives to forma syrup. An elixir is prepared by using a hydroalcoholic vehicle withsuitable sweeteners together with a flavouring agent. Suspensions can beprepared of the insoluble forms with a suitable vehicle with the aid ofa suspending agent such as acacia, tragacanth, methylcellulose.

[0069] For parenteral administration, fluid unit dosage forms areprepared utilizing an active ingredient and a sterile vehicle with waterbeing preferred. The active ingredient, depending on the form andconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the water-soluble active ingredient can bedissolved in water for injection and filter sterilized before fillinginto a suitable vial or ampoule and sealing. Advantageously, adjuvantssuch as a local anesthetic, preservative and buffering agents can bedissolved in the vehicle. Parenteral suspensions are prepared insubstantially the same manner except that an active ingredient issuspended in the vehicle instead of being dissolved and sterilizationcannot be accomplished by filtration. The active ingredient can besterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of theactive ingredient.

[0070] In addition to oral and parenteral administration, the rectal andvaginal routes can be utilized. An active ingredient can be administeredby means of a suppository. A vehicle which has a melting point at aboutbody temperature or one that is readily soluble can be utilized. Forexample, cocoa butter and various polyethylene glycols (Carbowaxes) canserve as the vehicle.

[0071] For intranasal instillation, fluid unit dosage forms are preparedutilizing an active ingredient and a suitable pharmaceutical vehicle,water being preferred, or by dry powder for insufflation.

[0072] For use as aerosols the active ingredients can be packaged in apressurized aerosol container together with a gaseous or liquefiedpropellant, for example, dichlorodifluoromethane, carbon dioxide,nitrogen, propane, and the like, with the usual adjuvants such ascosolvents and wetting agents, as may be necessary or desirable.

[0073] The invention will now be described with reference to thefollowing examples. It will be appreciated that the examples areprovided by way of illustration of the invention only and are notintended in any way to be limiting.

EXAMPLE ONE: ISOLATION AND CHARACTERIZATION OF PELORUSIDE A

[0074] A. Isolation

[0075] Sponge specimens were collected in Pelorus Sound, South Island,New Zealand at depths of 7-15M. A single frozen specimen (170 g wetweight, NIWA # 95DBMYC 2-6) was cut into small segments and extractedwith methanol (2×600 mL) for 24 hr. The second and first methanolicextracts were passed through a glass column packed with 75 mL of SupelcoDiaion HP20® polystyrenedivinylbenzene beads pre-equilibrated with 50%methanol in water. The eluents were combined and passed through the samecolumn. The resulting eluent was diluted with 150 mL of water and passedthrough the column. Finally the testing element was diluted with 2800 mLof water and passed back through the same column. The column was thenwashed with 100 mL of water and eluted with 150 mL fractions of 1) 20%acetone in water, 2) 55% acetone in water, 3) 55% acetone in 0.2 MNH₄OH, and 4) 55% acetone in 0.2 M NH₄OH adjusted to pH 4.9 with aceticacid. Fraction 2 was diluted with 150 mL of water and passed through aglass column packed with 35 mL of HP20® pre-equilibrated with water. Thecolumn was washed with 50 mL of water and eluted with 100 mL of acetone.The acetone eluent was concentrated to dryness to yield 78.8 mg of aviscous brown oil. The resulting oil was dissolved in 25 mL of methanoland passed through a small glass column containing 250 mg of TosoHassAmberchrom®. The column eluent was diluted with 60 mL of water andpassed back through the column. The column was washed with 20 mL ofwater and the loaded Amberchrom® was transferred on top of a 20×1.5 cmAmberchrom® column pre-equilibrated with water. The column was elutedwith increasing concentrations of acetone in water in a stepped gradientfashion. The 32-34% acetone in water fractions were concentrated todryness to yield a colourless oil (2.2 mg). The 38-40% acetone in waterfractions were concentrated to dryness to yield mycalamide A (10.6 mg).The fourth fraction eluted from the original HP20 column at pH 4.0 wasdiluted with 150 mL of water, adjusted to pH 7.0 with NH₃, and passedthrough a glass column packed with 30 mL of HP20® pre-equilibrated withwater. The column was washed with 50 mL of water and eluted with 100 mLof acetone. The acetone eluent was concentrated to dryness to yield 38mg of a yellow oil. The oil was dissolved in 12 mL of methanol andpassed through 2.5 mL of amino bonded phase packing material. The eluentwas concentrated to dryness to yield 11.7 mg of pateamine.

[0076] B. CHARACTERIZATION of the Compound Present in 32-34% AcetoneFraction

[0077] The structure of the compound present in the 32-34% acetonefraction was determined to be as follows:

[0078] The compound has been termed Peloruside A (Formula III). The ¹Hand ¹³C NMR assignments of Peloruside A in CDCl₃ are summarized in Table1 below: TABLE 1 ¹H and ¹³C NMR Assignments of Peloruside A in CDCl₃ ¹³C¹H Position δ (ppm) mult δ (ppm) mult, J (Hz) 1 173.95 s 2 70.26 d 4.53s 3 78.27 d 4.22 dd (10.5, 5.5) 4a 32.59 t 1.78 M 4b 2.13 m 5 63.51 d4.25 tdd (11, 4.5, 2.5) 6a 31.65 t 1.53 q (12) 6b 1.78 ddd (12.5, 5.5,2.5) 7 75.90 d 3.82 ddd (11.5, 5, 3) 8 66.84 d 4.02 d (3) 9 101.89 s 1043.63 s 11 73.85 d 4.89 br d (10) 12a 33.93 t 1.40 d (14.5) 12b 2.07 ddd(15, 11.5, 4.5) 13 77.88 d 3.99 brd (9.5) 14a 35.68 t 2.02 ddd (15.5,11.5, 1) 14b 2.15 555 (15.5, 10.5, 1) 15 70.86 d 5.68 d (10.5) 16 136.05s 17 131.13 d 5.05 d (10) 18 43.29 d 2.61 m 19a 24.60 t 1.17 m 19b 1.44m 20 12.23 q 0.85 t (7.5) 21 15.77 q 1.08 s 22 20.77 q 1.12 s 23 17.45 q1.67 d (1) 24a 66.94 t 3.36 t (10.5) 24b 3.64 dd (10.5, 4) 3Me 56.09 q3.31 s 7Me 55.68 q 3.38 s 13Me 59.06 q 3.48 s 6OH 6.75 s

[0079]FIG. 1 shows the ¹H NMR spectral signature of Peloruside A (300MHz; pulse sequence: s2pul).

[0080]FIG. 2 shows the IR spectral signature of Peloruside A.

[0081] C. Bioactivity of Peloruside A

[0082] Part 1

[0083] The bioactivity of peloruside A as an anti-tumor agent wasdetermined by an anti-tumor assay. For the anti-tumor assay a 2-folddilution series of the same of interest is incubated for 72 hours withP388 (Murine Leukemia) cells. The concentration of sample required toreduce the P388 cell growth by 50% (comparative to control cells) isdetermined using the absorbance values obtained with the yellow dye MTTtetrazollum is reduced by healthy cells to the purple colour MTTformazan. The result is expressed as an IC₅₀ in ng/mL.

[0084] Results/Conclusion

[0085] Peloruside A was found to be cytotoxic to P388 murine leukemiacells at approximately 10 ng/mL. Although it bears some structuralfeatures of both mycalamide A (gem-dimethyls and poly-hydroxylation) andpateamine (macrolide ring), it is not closely related biochemically.

[0086] Part 2

[0087] Cytotoxicity Assays

[0088] The cytotoxicity of Peloruside A was tested in five cell lines:

[0089] LLC-PK1 (pig kidney)

[0090] H441 (human lung adenocarcinoma)

[0091] SH-SY5Y (human neuroblastoma)

[0092] P388 (murine leukemia)

[0093] 32D (murine myeloid) generally in accordance with the MTT assayof Burres et al., J. Cancer Research (1989), 49, 2935-2940.

[0094] Briefly, cell lines were maintained in Dulbecco's modifiedEagle's medium: F12 medium (50:50) (Gibco) supplemented with 10% fetalcalf serum (Gibco), 100 mg/mL Penicillin G, and 50 mg/mL streptomycinsulfate. After 96 hours exposure to the toxin, cell viability wasdetermined by the MTT calorimetric assay. MTT standard curves weredetermined for each cell line, and the MTT absorbance over a range ofcell densities was found to be linear for each. Data were analysed withthe SYSTAT statistical program using a non-linear model fit, and IC50values were calculated using a Logit-Log plot.

[0095] The following results (expressed as LD₅₀ in nM) were obtained:Cell Line: LD50 (nM): P388 18 H441 6.2 LLC-PK1 3.7 SH-SY5Y 14.9 32D 7.8

[0096] Additional Observations

[0097] In cell line H441, the nuclei of the cells were observed to breakup into small vessicles (nuclear blebbing). This has not been observedfor mycalamide A and pateamine at their respective LD50s.

[0098] In cell line SH-SY5Y no retraction of dendrites was observedwhich contrasts with what has been observed with mycalamide A andpateamine.

[0099] In cell line 32D the LD50 was found to increase to 1.6 mM whenthe cells were assayed for viability at 24 hours of exposure. Thisdramatic increase in LD50 has not been observed for mycalamide A,pateamine cyclohexamide.

[0100] Conclusion

[0101] These results and observations confirm Peloruside A to be apotent cytotoxin. In particular, the results of the assay conducted inrelation to cancerous cell lines are predictive of anti-tumor efficacyin mammals, including humans.

EXAMPLE TWO: ACTIVITY OF PELORUSIDE A

[0102] A. Materials and Methods

[0103] Materials

[0104] Peloruside A was isolated as above and was stored at −20° C. as a1 mM solution in absolute ethanol. Paclitaxel, purified tubulin, andmouse monoclonal anti-rat b-tubulin were purchased from Sigma ChemicalCo. (St. Louis, Mo.).

[0105] Preparation of the Reduction Product of Peloruside A

[0106] NaBH₄ (2.5 mg) was added to a solution of Peloruside A (1 mg/1.5mL MeOH). After 12 h, the reaction was quenched with H₂O (4 mL) andpassed through an Amberchrom column (1×2 cm). The eluent was thendiluted with H₂O (4 mL) and passed through the column. The eluent wasdiluted with H₂O (8 mL) and passed through the column again. The columnwas eluted with H₂O (4 mL) and then MeOH (3 mL). The MeOH fraction wasconcentrated to dryness under vacuum to give the reduction product (0.8mg).

[0107] Less than 2% of the parent compound remained in the sample afterthe reduction.

[0108] Cell Culture and Cytotoxicity

[0109] Tumorigenic and non-tumorigenic cell lines were cultured aspreviously described in Hood K. A., West L. M., Northcote P. T.,Berridge M. V., Miller J. H., Apoptosis (2001), 6, 207-219. These celllines included HL-60 and KS62, two tumorigenic human myeloid leukemiccell lines, 32D clone 3 (32D), a non-tumorigenic murine myeloid cellline, 32D-ras, the ras-transformed derivative of 32D, H441, a human lungadenocarcinoma cell line, SH-SY5Y, a human neuroblastoma cell line, andLLC-PK₁, a non-tumorigenic pig kidney cell line. IC₅₀ values forPeloruside A in the different cell lines were determined using thetetrazolium-based MTT cell proliferation assay as previously described(Hood K. A et al.).

[0110] Anti-inflammatory and Metabolic Activity

[0111] The effect of Peloruside A and paclitaxel on superoxideproduction was determined using human peripheral blood neutrophilsactivated with 1 mM N-formyl-met-leu-phe (fMLP) as described previously(Tan A. S., Berridge M. V., J. Immunol. Meth. (2000), 238, 59-68). Inthis microplate assay, the cell-impermeable tetrazolium salt, WST-1, isreduced to its soluble formazan and dye reduction measured at 450 nm asan initial rate over 10-20 min. Samples were equilibrated with cells for3 min at 37° C. and the reaction initiated by adding fMLP. Measurementof anti-metabolic activity followed a similar microplate protocol exceptthat HeLa cells were used instead of neutrophils, cells were notactivated with fMLP, and the intermediate electron acceptor, 1-methoxyphenazine methosulfate at 25 mM, was included in the WST-1 reagent tofacilitate detection of low potential electrons from the plasmamembrane.

[0112] Flow Cytometry

[0113] Using standard methodology, the DNA of cells was stained withpropidium iodide (PI), and the proportion of cells in different phasesof the cell cycle was monitored by flow cytometry. Briefly, H441 cellswere treated with 1 mM Peloruside A or 1 mM paclitaxel for 24 h.Adherent cells were collected by trypsinization and added to those insuspension. The cells were then fixed with cold 70% ethanol overnightand stained with PI solution consisting of 45 mg/mL PI, 10 mg/mL RNaseA,and 0.1% glucose. After a 2 h incubation at RT, samples were analysed ina FACSort flow cytometer (Becton Dickinson).

[0114]FIG. 4 shows morphological changes in H441 cells. Phase-contrastphotomicrographs of H441 human lung adenocarcinoma cells: untreatedcontrol cells (A) and cells exposed for 48 h to 100 nM Peloruside A (B)or 100 nM paclitaxel (C). Note the intracellular fiber bundles inapproximately 10% of the treated cells and the numerous micronuclei,each with a dark central spot of condensed DNA. Scale bar=50 mm.

[0115]FIG. 5 shows Peloruside A-induced G₂/M cell cycle arrest. Cellswere treated with 1 mM Peloruside A or paclitaxel, stained with PI, andcounted by flow cytometry. For the mitotic index, cells were treated for24 h with different concentrations of Peloruside A or paclitaxel, andthe number of cells in mitosis divided by the total number of cells(n=at least 400 cells counted in each of 3 preparations).

[0116] In Situ Tubulin Polymerization

[0117] A simple in situ cellular assay as described by Giannakakou P.,Gussio R., Nogales E., Downing K. H., Zaharevitz D., Bollbuck B., PoyG., Sackett D., Nicolaou K. C., Fojo T., Proc. Nat. Acad. Sci. (USA)(2000), 97, 2904-2909 was used in which the shift in tubulin fromdepolymerized to polymerized forms was followed by electrophoresis andWestern blotting of centrifuged particulate and cytosolic fractions. Tosummarize, 2×10⁶ untreated and drug-exposed HL-60 cells were lysed byexposure for 5 min at 37° C. to 100 mL of hypotonic buffer (1 mM MgCl₂,2 mM EGTA, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 1 mg/mLaprotinin, 2 mg/mL pepstatin, and 20 mM Tris-HCl, pH 6.8) and theparticulate fraction separated from the soluble cytosolic fraction byhigh speed centrifugation for 10 min in a bench-top centrifuge. Sampleslabeled ‘0 min’ received drug immediately before collection of thecells. The processing of the cells to the critical centrifugation steprequired approximately 30 min. The pellet was dissolved in 100 mL ofsample buffer (8 M urea, 4% CHAPS, 3 M thiourea, and 40 mM DTT). TwentymL of loading buffer was added to each 100 mL sample, the samples werevortexed and then boiled for 5 min. Twenty mL of each sample was loadedon an SDS/10% polyacrylamide gel and resolved by electrophoresis.b-Tubulin bands were identified by Western blotting using b-tubulinprimary antibody (1/1000 dilution) following standard immunoblottingprocedures with detection by enhanced chemiluminescence (Lumi-Light,Roche).

[0118] In Vitro Tubulin Polymerization and Electron Microscopy

[0119] Purified tubulin (approximately 7.5 mg protein) containingapproximately 15% microtubule-associated proteins was obtained fromSigma and reconstituted in 0.1 M MES buffer, pH 6.8, 1 mM EGTA, 0.1 mMEDTA, 0.5 mM MgCl₂, 1 mM DTT, 0.1 mM GTP, 1 mg/mL leupeptin, 1 mg/mLaprotinin, and 100 mg/mL sucrose as stabilizer. The reconstitutedtubulin was incubated at 37° C. for 30 min in the presence of 10 mMPeloruside A or 10 mM paclitaxel. Samples (2 mL) were pipetted onto400-mesh carbon- and Formvar-coated copper grids and left for 2 min atRT before blotting with filter paper. Each grid was stained with 5 mL of1% uranyl acetate for 3 min at RT, then blotted with filter paper. Gridswere air-dried overnight before examination in a Philips CM100transmission electron microscope.

[0120]FIG. 6 shows Peloruside A-induced tubulin polymerization: (A)Immunoblots of b-tubulin following electrophoresis of soluble (S) andparticulate (P) fractions of HL-60 cells treated with differentconcentrations of drug for 5 h. (B) Immunoblots following exposure to 1mM Peloruside A and 1 mM paclitaxel for varying lengths of time. (C)Transmission electron micrograph of microtubules formed followingtreatment of purified soluble tubulin with 10 mM Peloruside A for 30 minat 37° C. Scale bar (lower right)=500 nm.

[0121] B. Results

[0122] Chemical Structures. The macrolide ring structures of PelorusideA, paclitaxel, epothilone B, and laulimalide are compared in FIG. 3,along with the structure of the reduction product of Peloruside A inwhich the 6-membered pyranose ring is opened by chemical reduction,generating a secondary alcohol at C₉.

[0123] Cytotoxicity. IC₅₀ values following a 4-day exposure toPeloruside A ranged from 4-15 nM in the different cell lines. No cleardifferences in MTT response were observed between tumorigenic cell lines(H441, SY5Y, HL-60, 32D-ras) and non-tumorigenic cell lines (32D,LLC-PK₁). The IC₅₀ values for Peloruside A (7±4 (S.E.M.) nM) andpaclitaxel (22±8) were similar in HL-60 cells. Reduction of Peloruside Awith NaBH₄ increased its IC₅₀ value 31 -fold in HL-60 cells (221±24 nM).

[0124] Cellular Morphology. After 2 days exposure to 100 nM Peloruside Aor paclitaxel, multiple micronuclei were observed in H441 cells (FIGS.4B, C) and K562 cells (data not presented). Longer exposures increasedthe number of micronuclei and the number of cells containingmicronuclei. The center of the micronuclei stained strongly with PI,indicating double-stranded DNA was present (data not presented). In bothPeloruside A- and paclitaxel-treated H441 cells, large intracellularfiber bundles were observed by phase-contrast microscopy (FIGS. 4B, C).

[0125] Anti-inflammatory and Metabolic Activity. Peloruside A andpaclitaxel were tested for their ability to inhibit the fMLP-activatedrespiratory burst of human neutrophils in vitro. At high concentrationsof Peloruside A (26 mM), 26% inhibition of superoxide production wasobserved whereas paclitaxel (12 mM) had no effect on neutrophilactivation. In a similar short-term assay that measures metabolicactivity in proliferating cells, Peloruside A at 180 mM stimulated WST-1 reduction by 20%, rather than inhibiting as might be expected for apotent cytotoxic agent. In this assay, paclitaxel inhibited WST-1reduction by 70% at 120 mM but had little effect at 12-24 mM.

[0126] Cell Cycle Arrest. Treatment of H441 cells with 1 mM Peloruside Aor paclitaxel for 24 h led to partial cell cycle arrest at G₂/M (FIG.5). The progression of cells into apoptosis/necrosis was seen as anincrease in the number of cells in the subdiploid peak. The arrest inG₂/M was more complete for paclitaxel than for Peloruside A, and thisdifference was mirrored in the mitotic index of the cultures. Cellstreated with 1 mM Peloruside A had 34±2% metaphase-arrested cellswhereas 64±4% of cells showed metaphase arrest following paclitaxeltreatment. Control H441 cultures without drug typically had about 4%cells in mitosis (FIG. 5).

[0127] Tubulin Polymerization. Tubulin in soluble and particulatefractions from HL-60 cells exposed to different concentrations ofPeloruside A, paclitaxel, Peloruside A reduction product, or colchicinefor 5 h were isolated and visualized by immunoblotting for b-tubulin(FIG. 6A). Peloruside A and paclitaxel caused similar, dose-dependentshifts of soluble tubulin to the particulate fraction. No detectableb-tubulin remained in the soluble fraction at 100 nM of either drug.Peloruside A reduction product had no significant effect on theproportion of soluble to polymerized tubulin in HL-60 cells. Colchicine,as expected, caused depolymerization of tubulin, with most of thetubulin in the soluble fraction at 1 mM concentration of drug.

[0128] A 20 min time course was carried out in the presence of 1 mMPeloruside A and 1 mM paclitaxel (FIG. 6B). By 5 min, both Peloruside Aand paclitaxel had converted almost all detectable tubulin to thepolymerized form.

[0129] 10 mM Peloruside A, like paclitaxel, caused purified tubulin topolymerize in solution into typical, long, straight microtubules at 37°C. (FIG. 6C). In the absence of drug, only a few sparse microtubuleswere seen by electron microscopy. Once formed, the microtubules inducedby Peloruside A and paclitaxel were stable at 0C.

[0130] C. Discussion

[0131] Peloruside A alters microtubule dynamics in a manner similar tothat reported for paclitaxel by inducing tubulin polymerization in situand in cell-free systems, causing cells to arrest in the G₂/M phase ofthe cell cycle. Despite the similarity of the primary mode of action ofPeloruside A to the taxanes, epothilones, and laulimalides, thestructure and some bioactivities of Peloruside A are unique, includingits anti-inflammatory activity and its possible enhancing effects oncell metabolism. In addition, Peloruside A was less effective thanpaclitaxel at causing mitotic arrest in H441 cells. These uniqueproperties present novel benefits for anti-cancer targeting. Based onthin layer chromatography results, Peloruside A is less lipophilic thanpaclitaxel, and this property should aid the clinical application ofPeloruside A or its analogues, since some of the side-effects ofpaclitaxel relate to its low aqueous solubility (6-11 mM) (Ter Haar E.et al.). Discodermolide is estimated to be 160-fold more soluble thanpaclitaxel, based on an indirect, fragment-based computationalcalculation (Ter Haar E. et al.). Laulimalides presumably have lowaqueous solubility since they were selected in part on the basis oftheir lipophilic properties (He L. et al. and Mooberry S. L. et al.).

[0132] Peloruside A induced the formation of multiple micronuclei,intracellular bundles, and metaphase arrest in a manner similar topaclitaxel, epothilone, and laulimalide. Cell-type specific differencesexist in the reported responses to paclitaxel, since some cells, such asHL-60 and the colon carcinoma cell line HT-29 arrest in metaphase, thenundergo apoptosis, whereas other cells, such as K562 and the melanomacell line SK-MEL-28, progress through metaphase and become polyploid inthe presence of drug (Banerjee S., Fallis A. G., Brown D. L., Oncol.Res. (1997), 9, 237-248 and Roberts J. R., Allison D. C., Donehower R.C., Rowinsky E. K., Cancer Res. (1990), 50, 710-716). The apoptosisinduced by Peloruside A (Hood K. A. et al.) is presumed to be aconsequence of G₂/M block or the DNA damage due to abnormal mitoticarrest. Mitotic arrest often induces apoptosis in cultured cells (BollagD. M. et al., and Wang T. H., Wang H. S., Soong Y. K., Cancer (2000),88, 2619-2628). With epothilone and paclitaxel, endonucleolytic cleavageof DNA, measured by the TUNEL assay, is only seen in G₂/M-blocked cells(Bollag D. M. et al, and Wang T. H. et al). The apoptotic pathway forpaclitaxel has been directly examined.

[0133] The evidence that Peloruside A is a microtubule-stabilizing agentis based on an in situ cell assay (FIGS. 6A, B) and an in vitropolymerization assay (FIG. 6C) in which a shift in tubulin from asoluble to a particulate form was observed. This conclusion thatPeloruside A stabilizes microtubules in a manner similar to the taxanesand other microtubule-stabilizing drugs is also supported by the G₂/Mcell cycle arrest data of FIG. 5. More direct measurements of PelorusideA-tubulin interactions in cell-free systems will be needed to fullydescribe the primary mode of action of Peloruside A, and theseexperiments are in progress.

[0134] Peloruside A is a novel natural product which, together with itsfunctionally equivalent analogues, has paclitaxel-likemicrotubule-stabilizing activity. Peloruside A and its functionallyequivalent analogues represent new drugs in an elite group of drugs ofmajor importance in the clinical treatment of solid tumors. Pelorutide Ais structurally distinct and may present a unique profile of bioactivitythat will add to that of the limited number of other knownmicrotubule-stabilizing drugs available for development.

[0135] INDUSTRIAL APPLICATION

[0136] Thus, in accordance with the present invention, the applicantsprovide a new bioactive compound, and its functionally equivalentanalogues, which have cytotoxic properties. These compounds can beformulated into medicaments, including pharmaceutical compositions, foruse in any prophylactic or therapeutic application for which theircytotoxic properties make them appropriate. Such therapeuticapplications include anti-tumor treatment.

[0137] Those persons skilled in the art will appreciate that the abovedescription is provided by way of example only and that variations andmodifications can be made without departing from the scope of theinvention which has been made.

1. A compound of formula (I);

wherein R₁, R₂, R₃, R₄ and R₅ are independently hydrogen, alkyl or acyl;or a functionally equivalent analogue thereof; or a compound of formula(II);

wherein X is O or ═C(R₅)R₆ and R₁, R₂, R₃, R₄, R₅, and R₆ areindependently hydrogen, alkyl or acyl; or a functionally equivalentanalogue thereof.
 2. A compound of formula (I) as defined in claim 1 ora functionally equivalent analogue thereof.
 3. A compound of formula(II) as defined in claim 1 or a functionally equivalent analoguethereof.
 4. A compound according to claim 1 of formula (III);

or a functionally equivalent analogue thereof.
 5. A compound of formula(IV);

or a functionally equivalent analogue thereof.
 6. A bioactive compoundwhich has the NMR and/or IR spectral signature of FIGS. 1 and
 2. 7. Acomposition which comprises a compound of formula (I) or formula (II) asdefined in claim 1, or a functionally equivalent analogue thereof,together with a suitable carrier therefor.
 8. A pharmaceuticalcomposition which comprises a compound of formula (I) or formula (II) asdefined in claim 1, or a functionally equivalent analogue thereof,together with a pharmaceutically-acceptable carrier therefor.
 9. Apharmaceutical composition which comprises a compound of formula (III)as defined in claim 4, or a functionally equivalent analogue thereof,together with a pharmaceutically-acceptable carrier therefor.
 10. Apharmaceutical composition according to claim 9 wherein the compound isof formula (III) as defined in claim
 4. 11. A method of prophylaxis ortherapy which comprises the step of administering to a patient in needof the same a compound of formula (I) or formula (II) as defined inclaim 1, or a functionally equivalent analogue thereof.
 12. The methodof claim 11 wherein the prophylaxis or therapy is achieved by inhibitingthe proliferation of cells in a mammal.
 13. The method of claim 12wherein the cells are tumor cells.
 14. The method of claim 11 whereinthe compound is administered in an amount effective to providemicrotubule stabilization.
 15. The method of claim 11 wherein theprophylaxis or therapy is of a pathological condition caused by theproliferation of cells in a mammal.
 16. The method of claim 15 whereinthe cells are tumor cells.
 17. The method of claim 11 which is, or ispart of, a treatment against cancer.
 18. A method of prophylaxis ortherapy which comprises the step of administering to a patient in needof the same a compound of formula (III) as defined in claim 4, or afunctionally equivalent analogue thereof.
 19. The method of claim 18wherein the prophylaxis or therapy is achieved by inhibiting theproliferation of cells in a mammal.
 20. The method of claim 19 whereinthe cells are tumor cells.
 21. The method of claim 18 wherein thecompound is administered in an amount effective to provide microtubulestabilization.
 22. The method of claim 18 wherein the prophylaxis ortherapy is of a pathological condition caused by the proliferation ofcells in a mammal.
 23. The method of claim 22 wherein the cells aretumor cells.
 24. The method of claim 18 which is, or is part of, atreatment against cancer.
 25. A method of prophylaxis or therapyaccording to claim 18 wherein the compound is of formula (III) asdefined in claim
 4. 26. A method of prophylaxis or therapy whichcomprises the step of administering to a patient in need of the same acomposition according to claim
 7. 27. A method of prophylaxis or therapywhich comprises the step of administering to a patient in need of thesame a pharmaceutical composition according to claim
 8. 28. A method ofprophylaxis or therapy which comprises the step of administering to apatient in need of the same a pharmaceutical composition according toclaim
 9. 29. A method of prophylaxis or therapy which comprises the stepof administering to a patient in need of the same a pharmaceuticalcomposition according to claim 10.